The following discussion of the background is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
There are, beside antibodies, novel binding proteins or binding domains that can be used to specifically bind a target molecule (e.g. Binz, H. K., Amstutz, P., Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005). One such novel class of binding proteins or binding domains not possessing an Fc are based on designed repeat proteins or designed repeat domains, such as designed ankyrin repeat proteins or designed ankyrin repeat domains (WO 2002/020565; Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Gruner, M. G., Pluckthun, A., Nat. Biotechnol. 22, 575-582, 2004). WO 2002/020565 describes how large libraries of repeat proteins, such as ankyrin repeat proteins, can be constructed, and their general application. WO 2012/069654 describes recombinant binding proteins comprising a designed ankyrin repeat domain with binding specificity for serum albumin. WO 2010/060748 describes recombinant binding proteins comprising designed ankyrin repeat domains with binding specificity for VEGF-A, and WO 2011/135067 describes modified versions of such recombinant binding proteins specific for binding to VEGF-A. WO 2014/191574 describes recombinant binding proteins comprising designed ankyrin repeat domains with binding specificity for HGF. None of these patent applications discloses a recombinant binding protein comprising a designed ankyrin repeat domain with binding specificity for VEGF-A and a designed ankyrin repeat domain with binding specificity for HGF.
Unlike e.g. IgG antibodies, which exhibit long systemic half-lives mediated by FcRn recycling, proteins comprising designed ankyrin repeat domains typically exhibit a fast pharmacokinetic clearance and short terminal half-lives, unless the protein comprises elements that improve the pharmacokinetic properties, such as e.g. a designed ankyrin repeat domain with binding specificity to serum albumin described in WO 2012/069654. Using serum albumin binding for improving pharmacokinetic properties of proteins is a process well-known in the art (see e.g. WO 9101743; Frejd F. Y., 2012 (in Kontermann, R (Ed.) “Therapeutic proteins: strategies to modulate their plasma half-lives”, Wiley-VCH Verlag GmbH, 2012, ISBN 978-3-527-32849-9); Nguyen, A., Reyes, A. E. II., Zhang, M., McDonald, P., Wong, W. L., Damico, L. A., Dennis, M. S. Protein Eng. Des. Sel. 19, 291-297, 2006; WO 2008/096158; WO 2006/122787; WO 2011/095545; and WO 2012/069654). In order to be able to use designed ankyrin repeat domains with binding specificity for serum albumin in clinical drug candidates, the storage stability of known designed ankyrin repeat domains with binding specificity for serum albumin has to be improved. Disclosed herein are designed ankyrin repeat domains with binding specificity for serum albumin with improved properties.
The effect of valency of designed ankyrin repeat domains with binding specificity for serum albumin on the pharmacokinetic properties of recombinant binding proteins has not been investigated. Based on findings of the albumin binding domain (Hopp, J., Horning, N., Zettlitz, K. A., Schwarz, A., Fuss, N., Müller, D., Kontermann, R. E. Protein Eng. Des. Sel. 23, 827-834, 2010), one skilled in the art would expect that a recombinant binding protein comprising two albumin binding protein domains such as designed ankyrin repeat domains with binding specificity for serum albumin would not have improved pharmacokinetic properties compared to a recombinant binding protein comprising only one designed ankyrin repeat domain with binding specificity for serum albumin. Surprisingly, we found that this is not the case. Disclosed are thus recombinant binding proteins comprising at least two designed ankyrin repeat domains with binding specificity for serum albumin that exhibit improved pharmacokinetic properties (i.e. prolonged terminal half-lives, increased exposures, reduced clearance, and/or increased percentages of injected dose) compared to recombinant binding proteins comprising only one designed ankyrin repeat domain with binding specificity for serum albumin.
Neovascularisation (new blood vessel formation) is widely known to play an important role in the development and maintenance of tumors (Ferrera, N., and Kerbel, R. S., Nature 438, 967-974, 2005). Accordingly, the inhibition of angiogenesis has become a main cornerstone in modern clinical oncology; especially the targeting of vascular endothelial growth factor (VEGF) and its receptors (Hurwitz, H., Clin. Colorectal Cancer, Suppl. 2, 62-68, 2004; Escudier, B., Clin. Adv. Hematol. Oncol. 5, 530-531, 2007). The mammalian VEGF family consists of five glycoproteins referred to as VEGF-A, VEGF-B, VEGF-C, VEGF-D (also known as FIGF) and placenta growth factor (PIGF, also known as PGF). VEGF-A has been shown to be an effective target for anti-angiogenic therapy (Weis, S. M., and Cheresh, D. A., Nat. Med. 17, 1359-1370, 2011). The VEGF-A ligands bind to and activate three structurally similar type III receptor tyrosine kinases, designated VEGFR-1 (also known as FLT1), VEGFR-2 (also known as KDR) and VEGFR-3 (also known as FLT4). Several angiogenesis inhibitors have received regulatory approval to date showing a prolonged progression-free survival (PFS) and/or overall survival in various cancer types in combination with chemotherapy. Unfortunately, resistance inevitably occurs during the course of treatment with VEGF/VEGFR inhibitors, such as the VEGF-A inhibitor bevacizumab (Avastin®), suggesting that concomitant inhibition of additional targets and resistance pathways may be necessary to achieve superior clinical results (Kerbel, R. S., N. Engl. J. Med. 358, 2039-2049, 2008; Hurwitz, 2004, loc. cit.; Escudier, 2007, loc. cit.). cMet tyrosine kinase is a cell surface receptor for hepatocyte growth factor (HGF, also known as scatter factor, SF) primarily expressed on epithelial cells (Comoglio, P. M., Giordano, S., and Trusolino, L., Nat. Rev. Drug Discov. 7, 504-516, 2008). While cMet and HGF are expressed at low levels in normal adult tissues, their expression is frequently up regulated in a broad range of human tumors, which has been correlated in preclinical models with tumor cell survival, growth, angiogenesis, invasion and metastasis (Rong, S., Segal, S., Anver, M., Resau, J. H., Vande Woude, G. F., Proc. Natl. Acad. Sci. USA 91, 4731-4735, 1994; Michieli, P., Mazzone, M., Basilico, C., Cavassa, S., Sottile, A., Naldini, L., Comoglio, P. M., Cancer Cell 6, 61-73, 2004). Up-regulation of HGF and/or cMet expression and signaling has been found to be associated with poor prognosis and drug resistance in many tumor types in the clinic (Fasolo, A., Sessa, C., Gianni, L., Broggini, M., Ann. Oncol. 24, 14-20, 2013). Altogether this indicates that the HGF-cMet axis is an important target for therapeutic intervention (Comoglio, 2008 loc. cit.; Fasolo et al., 2013, loc. cit.). Through binding to its receptor, HGF mediates a number of cellular responses, including scattering of various cell types, the formation of tubules and lumens, epithelial-mesenchymal transition, angiogenesis, liver regeneration, wound healing and embryological development. The HGF/c-Met signaling pathway has also been shown to play a role in various diseases, including many human solid tumors, in which it participates in tumor development, invasion and metastasis. Current HGF/cMet pathway inhibitors in phase II or III clinical development comprise monoclonal antibodies (mAbs) targeting the extracellular domain of cMet (i.e. MetMab from Genentech-Roche) or small molecule inhibitors of its intracellular kinase domain. Small molecule inhibitors such as tivantinib (ArQule®) and cabozantinib (Cometriq®) are very potent but less specific than mAbs and bear the potential for higher toxicity. Biological agents against HGF/SF include rilotumumab (AMG102), a humanised mAb against HGF, and ficlatuzumab (AV-299), a humanised anti-HGF IgG1. The use of HGF/cMet inhibitors in combination with other targeted agents is an active field of investigation which aims to simultaneously inhibit various signaling pathways that have redundant or synergistic tumor functions. HGF/cMet triggers potent angiogenic signals that act synergistically with VEGF in inducing new tumor blood vessels and can induce resistance to anti-angiogenic therapy such as Avastin® and Sutent® (sunitinib) in glioblastoma (Jahangiri, A., De Lay, M., Miller, L. M., Carbonell, W. S., Hu, Y. L., Lu, K., Tom, M. W., Paquette, J., Tokuyasu, T. A., Tsao, S., Marshall, R., Perry, A., Bjorgan, K. M., Chaumeil, M. M., Ronen, S. M., Bergers, G., Aghi, M. K., Clin. Cancer Res. 19, 1773-1783, 2013) and renal cell cancer, respectively.
There are currently a number of anti-HGF/cMet compounds under investigation in combination with other targeted agents such as anti-VEGF receptor inhibitors, which have demonstrated a favorable safety profile in a variety of tumor types (Sharma, P. S., Sharma, R., Tyagi, T. Curr. Cancer Drug Targets. 11, 624-653, 2011). However, such combination therapy approaches imply that the patient must receive two separate treatments, each with a different safety profile, which may lead to increased undesirable toxicities, which in turn may limit the medical treatment options. Furthermore, different treatments may be subjected to different administration schemes, which could make the dosing more burdensome for the patient. Last but not least, the dosing of various agents simultaneously may significantly increase the costs associated to treatment and patient care.
One commercially available drug with dual cMet and VEGF inhibitory activity is cabozantinib (Cometriq®; a small molecule drug), an oral, multi-specific tyrosine kinase inhibitor targeting cMet and VEGFR 1-3 (in addition to RET, KIT, AXL and FLT3). Cabozantinib has validated the clinical approach of simultaneously inhibiting HGF and VEGF in tumors with a single agent (Yakes, F. M., Chen, J., Tan, J., Yamaguchi, K., Shi, Y., Yu, P., Qian, F., Chu, F., Bentzien, F., Cancilla, B., Orf, J., You, A., Laird, A. D., Engst, S., Lee, L., Lesch, J., Chou, Y. C., Joly, A. H., Mol. Cancer Ther. 10, 2298-2308, 2011; Castellone, M. D., Carlomagno, F., Salvatore, G., Santoro, M., Best Pract. Res. Clin. Endocrinol. Metab. 22, 1023-1038, 2008). For instance, in castration resistant prostate cancer, an indication where the anti-HGF mAb rilotumumab failed to demonstrate efficacy as single agent in phase II studies, cabozantinib showed anti-tumor activity in a high percentage of patients in phase II (Smith, D. C., Smith, M. R., Sweeney, C., Elfiky, A. A., Logothetis, C., Corn, P. G., Vogelzang, N. J., Small, E. J., Harzstark, A. L., Gordon, M. S., Vaishampayan, U. N., Haas, N. B., Spira, A. I., Lara, P. N. Jr., Lin, C. C., Srinivas, S., Sella, A., Schöffski, P., Scheffold, C., Weitzman, A. L., Hussain, M., J. Clin. Oncol. 31, 412-419, 2013). However, activity was paralleled with a high incidence of adverse events that led to dose reductions in 62% of patients, raising doubts on the safety and tolerability of such pleiotropic modes of action.
Simultaneous targeting of VEGF-A and HGF/cMet may beneficially disrupt angiogenesis and tumor progression. As described hereinbefore, current therapies acting simultaneously on the VEGF-A/VEGFR-2 and the HGF/cMet-pathways either are based on single therapeutics that are unspecific and lead to safety findings, or involve several specific therapeutics that have to be combined, resulting in a need of co-administration or multiple administrations. Furthermore, some of the current drugs exhibit short systemic half-lives. Thus, there is a need to provide improved drugs blocking the VEGF-A/VEGFR-2 and the HGF/cMet pathways. This is technically difficult to achieve with antibody drugs, which further suffer from the need of laborious production in mammalian cells. Provided herein are recombinant binding proteins that address these issues. In some embodiments a recombinant binding protein provided herein comprises at least one designed ankyrin repeat domain with binding specificity for VEGF-A, at least one designed ankyrin repeat domain with binding specificity for HGF, and, for pharmacokinetic property improvement, at least two designed ankyrin repeat domains with binding specificity for serum albumin.